Supplementary Figure 6: Transgenic expression of Glut1 diminishes the suppressive ability of T_reg cells in vitro and in vivo.

a. CD4⁺CD25^- T cells were isolated from the spleens of control and Glut1-Tg mice and polarized under T_reg skewing conditions. Control and Glut1-Tg T_reg were functionally examined in an in vitro suppression assay to measure inhibition of effector T cells (T_eff) proliferation and the T_eff division index was calculated by Flowjo flow cytometry analysis software. b-d. RAG1^-/- mice were injected with naïve effector (CD4⁺CD25^-CD45RB^hi) T cells to induce colitis. After weight loss indicated active disease was apparent, control or Glut1-Tg CD4⁺CD25⁺CD45RB^lo T_reg were sorted and analyzed by flow cytometry to assess sorted T_reg. (b) The expression of CD25 and CD45Rb and (c) Foxp3 protein of sorted rescue T_reg are shown. (d) At the termination of the experiment Foxp3 levels were assessed on CD4⁺ gated T cells in the spleens of recipient animals. (e) Thy1.1 naïve effector (CD4⁺CD25^-CD45RB^hi) T cells were adoptively transferred into RAG1^-/- mice to initiate IBD. Thy1.2 control or Glut1-tg tT_reg (CD4⁺CD25⁺CD45RB^lo) T cells were sorted and injected after disease was apparent Foxp3 levels were then assessed by flow cytometry on adoptively transferred Thy1.1 effectors and Thy1.2 CD4 control and Glut1-tg T_reg from mesenteric lymph nodes and spleens. Data are the result of three independent experiments (a), representative of three independent experiments (b) or is representative of two independent experiments with at least 5 mice per group (c, d). Means and standard deviations are shown, * p<0.05. (f) Model of our findings. Our findings show that T_reg are metabolically heterogeneous and depend on activating and inflammatory signals as well as Foxp3 itself to coordinate metabolism. In the presence of inflammatory stimuli, such as TLR ligands, T_reg increase mTORC1 signaling, Glut1, and glycolysis, which results in increased cell growth and proliferation. Suppressive capacity, however, can be impaired. As inflammatory signals are reduced, Foxp3 can tilt the balance away from mTORC1 signaling to favor oxidative metabolism that lowers proliferative ability but enhances suppression to promote inflammatory resolution. Metabolic transitions are critical in this process as increased Glut1 expression is sufficient to promote T_reg growth while reducing suppression and stability.